Multi-allelic detection of SARS-associated coronavirus

ABSTRACT

The subject invention relates to a multiple-allelic RT-real-time polymerase chain reaction (PCR) assay for coronaviruses including the SARS virus. Multiple target sequences within the SARS-CoV, S, E, M and N genes are identified. The use of the four different targets enhances the likelihood that the fundamental genetic drift of the virus will not lead to a false negative result. Multiplex assays format for the assay are envisioned. Thus, the present invention allows for early diagnosis of a SARS infection. The assay would be useful in the context of monitoring treatment regimens, screening potential anti SARS agents, and similar applications requiring qualitative and quantitative determinations.

This application is the national stage filing of InternationalApplication No. PCT/US/2004/026380, filed Aug. 13, 2004 and claims thebenefit thereof. The International Application is based on U.S.Provisional Application No. 60/576,314, filed Jun. 3, 2004 and U.S.Provisional Application No. 60/496,995, filed Aug. 22, 2003.

BACKGROUND OF THE INVENTION

1. Field of Invention

This invention is directed to methods for the detection and/orquantitation of the SARS virus, reagents and test kits containing thesame for use in the method.

2. Background of the Invention

Severe acute respiratory syndrome (SARS) is one of the most recentemerging infectious diseases. The cause of SARS has been identified as anew coronavirus—a virus within the family Coronaviridae—designated asthe “SARS coronavirus” (SARS-CoV) [1, 2] by the World HealthOrganization, following assessment of causation according to Koch'spostulates, including monkey inoculation [3]. The coronaviruses areenveloped positive single-stranded RNA viruses with genomesapproximately 30 kb in length—the largest of any of the RNA viruses—thatreplicate in the cytoplasm of host cells without going through DNAintermediates. Coronaviruses have been reported to cause common colds inhumans, and to cause respiratory, enteric, and neurological diseases, aswell as hepatitis, in animals. Human coronaviruses are usually difficultto culture in vitro, whereas most animal coronaviruses and SARS-CoV caneasily be cultured in Vero E6 cells [4]. There are three groups ofcoronaviruses: Groups 1 and 2 encompass mammalian viruses, whereas Group3 encompasses avian viruses. Within each group, the coronaviruses areclassified into distinct species according to host range, antigenicrelationships, and genomic organization. Human coronaviruses (HCoVs)were previously reported to belong in Group 1 (HCoV-229E) and Group 2(HCoV-OC43), and are responsible for mild respiratory illnesses.

Recently, two independent groups, one at the British Columbia CancerAgency (BCCA) in Canada [5] (Tor2 isolate), and the other at the Centersfor Disease Control and Prevention (CDCP) in the United States [6](Urbani isolate), were first to obtain full genomic sequences ofSARS-CoV. Phylogenetic analyses, based on the genome sequences, revealedthat both isolates were distantly related to previously characterizedcoronaviruses, including the two previously isolated nonpathogenic humancoronaviruses strains, HCoV-C43 and HCoV-229E. The genome of the Tor2CoV isolate is 29,751 nucleotides long, and the genome of the Urbani CoVisolate is 29,727 nucleotides long, and their sequences differ at only24 nucleotide positions. The genomic organization of both isolates ischaracteristic of coronaviruses having the following typical gene order:5′-replicase (rep), spike (S), envelope (E), membrane (M), andnucleocapsid (N). The SARS-CoV rep gene, which is approximately 20,000nucleotides long, is predicted to encode two polyproteins (ORF1a andORF1b) that undergo proteolytic processing, resulting in severalnonstructural proteins. There are four genes downstream of rep thatencode the structural proteins S, E, M, and N.

The genome of SARS-CoV has several distinct genomic characteristics thatdistinguish it from other coronavirus isolates and that could be ofbiological significance. The gene encoding hemagglutinin-esterase, whichis present between ORF1a and S in Group 2 coronaviruses (and in someGroup 3 coronaviruses) is absent, and so is the short anchor of the Sprotein. Furthermore, the short anchor of the S protein, the specificnumber and location of the small ORFs, and the presence of only one copyof PLP^(PRO) provide a combination of genetic features that readilydistinguish SARS-CoV isolates from previously the describedcoronaviruses [5, 6]. There are several publications that describereverse-transcriptase polymerase chain reaction assays (RT-PCR assays)for the detection of SARS-CoV.

Perris et al. [2] developed as RT-PCR assay that identifies the virusfrom nasopharyngeal aspiration samples obtained from patients infectedwith SARS-CoA. Total RNA from clinical samples is reverse transcribed inthe presence of random hexamers, and the resulting cDNA is amplifiedwith primers 5′-TACACACCTCAGCGTTG-3′ (SEQ ID NO: 86) and5′-CACGAACGTGACGAAT-3′ (SEQ ID NO: 87). To determine the geneticsequence of an unknown RNA virus, they perform a random RT-PCR assay.Total RNA from virus-infected fetal rhesus kidney cells were isolated,reverse transcribed with primer 5′-GCCGGAGCTCTGCAGAATTCNNNNNN-3′ (SEQ IDNO: 88), and the resulting cDNA was amplified with primer5′-GCCGGAGCTCTGCAGAATTC-3′ (SEQ ID NO: 89).

Ksiazek et al. [1] developed a reverse transcription and real-time PCRassay to identify SARS-CoV. Oligonucleotide primers used foramplification and sequencing of the SARS-related coronavirus weredesigned from alignments in open reading frame 1b of the coronaviruspolymerase gene sequences. They used the primer pair IN-2 (+)5′-GGGTTGGGACTATCCTAAGTGTGA-3′ (SEQ ID NO: 90) and IN-4 (−)5′-TAACACACAACICCATCATCA-3′ (SEQ ID NO: 91), which was previouslydesigned to hybridize to conserved regions of the open reading frame 1b(ORF1b), in order to achieve broad reactivity with coronavirus/genus.These primers were used to amplify DNA from SARS isolates, and theamplicon sequences obtained were used to design SARS-specific primersCor-p-F2 (+) 5′-CTAACATGCRRAGGATAATGG-3′ (SEQ ID NO: 92), Cor-p-F3 (+)5′GCCTCTCTTGTTCTTGCTCGC-3′ (SEQ ID NO: 94), which were used in turn totest patient specimens. Drosten et al. [4] used a PCR-basedrandom-amplification procedure to genetically characterized a300-nucleotide-long SARS-CoV genomic segment. On the basis of thesequence that was obtained, conventional real-time PCR assays forspecific detection SARS-CoV ORF1b were established. Poon et al. [7]developed an RT-real-time-PCR assay. Total RNA isolated from stoolspecimens from SARS-CoV-infected individuals is reverse transcribed withrandom hexamers and resulting can is amplified with primers coro35′-TACACACCTCAGCGTTG-3′ (SEQ ID NO: 86) and CORO4 5′-CACGAACGTGACGAAT-3′(SEQ ID NO: 87), which recognize a region of the viral polymerase gene.It is important to note that these authors acknowledges in theirpublication that the primers that they use in their assay cancross-react with the nonpathogenic human coronavirus strain HCoV-OC43.

SARS-specific PCR priers and diagnostic procedures were developed inseveral World Health Organization laboratories for the amplification ofa region of the open reading frame 1b of the SARS-CoV polymerase genesequence. These primers are currently being assessed to determine theirrelative performance and sensitivity with difference specimens obtainedat different times over the course of illness. Lipkin and Breise haveannounced they develop a PCR-based SARS diagnostic that detects aSARS-CoV gene that is present in multiple copies, but no furtherinformation is available in the literature.

Problems with the prior art that the current invention is designed tosolve. The main problems with current molecular diagnostic assays are:a) failure to consider the intrinsically polymorphic nature ofcoronaviruses, including the current SARS-CoV strains originated fromthe Tor2 and Urbani isolates—the ability of the virus to mutate andrecombine during the period of time it is within the infectedindividual, and during horizontal transmission; and b) failure toaccount for the possibility of continuous and/or multiple introductionof non non-genetically identical SARS-CoV strains into the humanpopulation.

A characteristic of RNA viruses is their high rate of genetic mutation,which leads to evolution of new viral strains, and is well-establishedmechanism by which viruses escape the immune system. Coronaviruses,including SARS-CoA, are quite sloppy when it comes to replicating theirgenetic material, producing one error for every 10,000 nucleotides thatthey copy, which is roughly the same error rate as occurs during thereplication of human immunodeficiency virus, HIV-1. Coronavirus RNApolymerase sometimes jumps between multiple copies of the viral genomethat are present in an infected cell. Therefore, each new genome isactually copied from several templates, reducing the chance that anygiven mutation will become well established in the viral population.Moreover, if one of these jumps is imprecise, a whole chunk of genomecan get skipped, resulting in the deletion of part of an important gene.The consequences can be dramatic, particularly if the change affects theprotein spikes that enable the virus to bind to the surface of thehost's cells. For example, in 1984 a new respiratory sickness appearedin European pig farms. It turned out to be a deletion mutant of acoronavirus that previously had infected piglets' stomachs 8. Itpossessed an altered spike protein that enabled the virus to infect adifferent cell type. Although the new disease was not generally lethal,it has since spread worldwide, and has complicated the diagnosis of thegut disease. Another example is the recent introduction of SARS-CoV intothe human population. It is likely that a genetic deletion may havehelped the SARS virus strains to make the transition from its animalreservoir to humans. Genetics analyses of the viral strains found inanimals for sale in the Southern Chinese markets indicated that theseSARS strains lacked 29 nucleotides in the gene encoding a protein ofunknown function and the protein product of this gene is attached to theinside of the virus's coat protein. Furthermore, in a recentpublication, full genome sequences of 14 isolates from SARS-CoV-infectedpatients in Singapore, Toronto, China and Hong Kong were compared, and14 mutations were revealed 9. In one respect, this finding may be viewedas indicating that SARS virus fails to mutate; however, this virus hasso far encountered little resistance from it new human hosts, and therehas, therefore, been little selective pressure to cause new mutants tobe retained. SARS-CoV will probably not remain as stable as it has beenso far. Our immune systems could force changes, similar to the changesthat frequently occur in flu viruses. In summary, we deem it prudent todevelop a new SARS-CoV diagnostic assay that accounts for thegenetically polymorphic nature of coronaviruses, including SARS-CoV.

SUMMARY OF THE INVENTION

The present invention includes a molecular-beacon-based multi-allelicRT-real-time-PCR assay for the detection of and discrimination betweenSARS-associated and other coronavirus isolates in clinical samples. Themain elements of the assay design are: a) mismatch-tolerant molecularbeacons; b) four sets of PCR primers for four different viral genes, andfour different molecular beacons (each labeled with the samefluorophore, and each specific for a different SARS-CoV gene); c) anexogenous RNA standard that is added to the sample that can bereverse-transcribed and amplified by one of the primer sets; and d) afifth molecular beacon that is labeled with a different fluorophore thatis specific for the exogenous RNA standard. The assay further includesRNA isolation from clinical samples (blood, tissue, sputum,nasopharyngeal aspiration samples, and others), reverse transcription,PCR amplification and simultaneous automated implicit detection in aspectrofluorometric thermal cycler that measures the fluorescenceintensity of each color during the annealing phase of each thermalcycle.

Multiple target sequences within the SARS-CoV S, E, M and N genes(Urbani and Tor2 strains) were identified. The S, M, and E genes encodestructural proteins that are present on the outside of the virus,whereas the N gene encodes a structural protein that is required forviral RNA packaging inside the virion. The principle underlying theselection of four target sequences that uniquely identify SARS-CoV(rather than only one target sequence) is that the use of four differenttargets enhances the likelihood that the fundamental genetic drift ofthe virus will not lead to a false negative result—that is, one hasbetter chance of hitting a moving target with a shotgun than with arifle. Thus, by detecting four different target alleles in the sameassay tube, and by using a single-fluorophore detection system, thedesign of the assay significantly minimizes the likelihood of missingthe presence of the SARS-CoV in a clinical sample due to the continuousviral evolution of the viral sequence. Moreover, by simultaneouslydetecting four different target sequences in the same assay tube, theintrinsic sensitivity of the assay is enhanced.

In order to identify the best target sequences within each viral genethat discriminate the SARS-Urbani and SARS-Tor2 strains from othernonpathogenic human and animal coronavirus strains, we used DNAalignments and phylogenetic analysis of available coronavirus genesequences deposited in GenBank. DNA sequences of SARS-CoV genes werecompared with those from reference viruses representing each species inthe three known groups of coronaviruses [group 1 (G1): human coronavirus229E (HCoV-229E), af304460; porcine epidemic diarrhea virus (PEDV),af353511; transmissible gastroenteritis virus (TGEV), aj271965; caninecoronavirus (CCoV), d13096; feline coronavirus (FCoV), ay204704; porcinerespiratory coronavirus (PRCoV), z24675;—Group 2 (G2): bovinecoronavirus (BCoV), af220295; murine hepatitis virus (MHV), af201929;human coronavirus OC43 (HCoV-OC43), m76373; porcine hemagglutinatingencephalomyelitis virus (HEV), ay078417; rat coronavirus (RtCoV),af207551; and—Group 3 (G3): infectious bronchitis virus (IBV), m95169].Sequence alignments were performed by CLUSTALW, which is a multiplesequence alignment tool that is commonly used in the bioinformaticscommunity. It produces global multiple sequence alignments through threemajor phases: a) pairwise alignment, b) guide-tree construction, and c)multiple alignment. The guide tree generated by CLUSTALW is an estimateof relationships between sequences that are much like those shown byphylogenetic trees.

The criteria for selecting SARS-CoV gene-specific PCR primers were basedon: a) the identification of genomic regions in SARS-CoV that, as aresult of an examination of the sequence alignments, showed the highestgenetic distance between SARS-CoV and other coronavirus strains; b)selection of primer sequences for amplification of the SARS-CoV targetsthat form primer-target hybrids whose theoretical melting temperaturemaximizes the ability of the primer to bind to the target even ifnucleotide substitutions are present (mismatch tolerance), and yetenable all of the primers to hybridize to their targets at the sametemperature in a multiplex assay (T_(m) approximately 60° C.); and c)selection of primer sequences that enable the amplicons containing eachof the four target sequences to be approximately the same (relativelyshort) length (approximately 100 nucleotides long).

The criteria for selecting the molecular beacon probe sequences, andtheir arm sequences, were based on: a) the identification ofapproximately 30-nucleotide-long regions in SARS-CoV (within theamplicons to be generated) that, as a result of an examination of thesequence alignments, showed the highest genetic distance betweenSARS-CoV and other coronavirus strains (with special emphasis on probetarget sequences that encompass gaps or deletions in the SARS-CoVsequence compared to the sequence of other coronaviruses); b) selectionof probe sequences that form probe-target hybrids whose theoreticalmelting temperature maximizes the ability of the probe to bind to thetarget sequence even if nucleotide substitutions are present (mismatchtolerance), and yet enable all of the probes to hybridize to theirtargets at the same temperature in a multiplex assay (Tm approximately63° C.); and c) selection of arm sequences that provide the same degreeof stability for the stem hybrids of all of the molecular beacon probes(stem T_(m) of approximately 70° C.).

The assays described herein can be performed in either a heterogeneousor homogeneous format. The reagents needed for performance of the assaycan be supplied in a kit format. The kit contains the detectantsnecessary for measuring two or more of the coronavirus genes S, E, M andN. It is recommended that the kit also contain an internal standard IPC.The reagents including the detectants can be separately packaged inindividual containers. The kits may also contain a substrate includingreaction tubes for performing an assay for a given sample. The kit mayalso contain additional reagents for performing amplification reactionsincluding PCR and also for sample pretreatment including those reagentnecessary to release and/or purify the coronavirus.

When there is a need to perform two or more different assays on the samesample, most of the time in a single vessel and at about the same time,a multiplex format can be utilized. Such formats are known in the art.Multiplex assays are typically used to determine simultaneously thepresence or concentratin of more than one molecule in the sample beinganalyzed, or alternatively, several characteristics of a singlemolecule.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the DNA Sequence Alignment of Coronavirus S Genes isolatedfrom different species (SEQ ID NOS 1-13, respectively in order ofappearance).

FIG. 2 shows the Phylogenetic Analysis of S Gene.

FIG. 3 shows molecular designs for SARS-Associated S Gene. Theoligonucleotides shown are SEQ ID NOS 14-18, and 15, respectively inorder of appearance. The sequences in the alignment are SEQ ID NOS 1-13,and the three primers shown above the alignment are SEQ ID NOS 16-18,all respectively in order of appearance.

FIG. 4 lists Molecular Designs for S Gene (SEQ ID NO: 14, 19, 15, 16,20, 17, 21, and 18, respectively in order of appearance).

FIG. 5 shows the DNA Sequence Alignment of Coronavirus E Genes isolatedfrom different Species (SEQ ID NOS 22-33, respectively in order ofappearance).

FIG. 6 shows the phylogenetic analysis of E Gene.

FIG. 7 shows molecular designs for SARS-Associated E Gene. Theoligonucleotides shown are SEQ ID NOS 34-38, and 35, respectively inorder of appearance. The sequences in the alignments are SEQ ID NOS22-33, and the three primers shown above the alignment are SEQ ID NOS36-38, all respectively in order of appearance.

FIG. 8 shows molecular designs for SARS-Associated E Gene (SEQ ID NOS34, 39, 35, 36, 40, 37, 41, and 38, respectively in order ofappearance).

FIG. 9 shows the DNA Sequence Alignment of Coronavirus M Genes isolatefrom difference species (SEQ ID NOS 42-54, respectively in order ofappearance).

FIG. 10 shows the phylogenetic analysis of M Gene

FIG. 11 shows the molecular designs for SARS-Associated M Gene. Theoligonucleotides shown are SEQ ID NOS 55-59, and 56, respectively inorder of appearance. The sequences in the alignment are SEQ ID NOS42-54, and the three primers shown above the alignment are SEQ ID NOS57-59, all respectively in order of appearance.

FIG. 12 lists molecular designs for M Gene (SEQ ID NOS 55, 60, 56, 57,61, 58, 62, and 59, respectively in order of appearance).

FIG. 13 shows the DNA Sequence Alignment of Coronavirus N Genes isolatefrom difference species (SEQ ID NOS 63-75, respectively in order ofappearance).

FIG. 14 shows the phylogenetic analysis of N Gene.

FIG. 15 shows the molecular designs for SARS-Associated N Gene. Theoligonucleotides shown are SEQ ID NOS 76-80, and 77, respectively inorder of appearance. The sequences shown in the alignment are SEQ ID NOS63-75, and the three primers shown above the alignment are SEQ ID NOS78-80, all respectively in order of appearance.

FIG. 16 list molecular designs for N Gene (SEQ ID NOS 76, 81, 77, 78,82, 79, 83, and 80, respectively in order of appearance).

FIG. 17 shows molecular designs for Internal Positive Control (IPC). Theoligonucleotides shown are SEQ ID NOS 84, 85, 16, 37, and 85,respectively in order of appearance.

FIG. 18 lists molecular designs for IPC (SEQ ID NOS 84, 85, 16, 20, 37,41, and 38, respectively in order of appearance).

FIG. 19 shows Molecular Beacon Melting Curves.

FIG. 20 shows Uniplex Real-time PCR Amplifications (SerialDilutions-Dynamic Range).

DETAIL DESCRIPTION OF THE INVENTION

The general steps for the assay involve the performance of the followingsteps:

Step 1. An RNA standard that can be reverse-transcribed and amplified byone of the primer sets is added to each sample prior to isolation of RNAfrom the sample. RNA is then extracted from each clinical sample(nasopharyngeal aspirations, stool samples, or whole blood, obtainedfrom patients suspected of being infected with SARS-CoV). RNA is thenpurified, using a QIAamp Viral RNA Kit (Qiagen, Inc., Valencia, Calif.),according to the manufacturer's instructions.

Step 2. The isolated RNA obtained from each clinical sample is thenreverse transcribed with four target-specific primers (S Gene, S-RT5′-AGGCTGTAAGAA-3′ (SEQ ID NO: 18); E-Gene, E-RT 5′-TATTGCAGCAGTAC-3′(SEQ ID NO: 38); M Gene, M-RT 5′-AAGCAACGAAGTAG-3′ (SEQ ID NO: 59); NGene, N-RT 5′-GCCTTCTTGTTAG-3′ (SEQ ID NO: 80; Internal PositiveControl, E-RT 5′-TATTGCAGCAGTAC-3′ (SEQ ID NO: 38)). Reversetranscription with SARS-CoV Viral RNA from patient samples is performedby adding 20 μL viral RNA to a mixture of 0.125 μM of (each)gene-specific primer and incubating at 80° C. for 5 minutes to denaturesecondary structures. The tubes are then immediately placed on ice forat least 2 minutes. Reverse transcription reactions consist of PCRbuffer II (Applied Biosystems), 3 mM MgCl₂, 0.5 mM (each) dNTP, 10 mMdithiothreitoal, 20 Units riboneclease inhibitor (Roche MolecularBiochemicals, Indianapolis, IN) and 80 Units Superscript II RibonucleaseH-reverse transcriptase (GibcoBRL) in a final volume of 40 μL. Thereactions are then incubated at 42° C. for 50 minutes, followed byinactivation at 70° C. for 15 minutes.

Step 3. Real-time PCR amplification of SARS-CoV cDNA is performed usingfour primer pairs and five molecular beacons in the same reaction, oneprimer pair for each viral gene (amplification of the internal positivecontrol is enabled by one of the primer pairs that was designed toenable the amplification of one of the for the viral gene targets). EachPCR reaction consists of 20 μL of cDNA products, 1× PCR buffer II(Applied Biosystems), 3.5 mM MgCl₂, 0.5 mM (each) dNTP, 0.4 μM of eachprimer, and 2.5 Units of AmpliTaq Gold DNA polymerase (AppliedBiosystems) in a final volume of 50 μL. Fifty cycles of amplification(94° C. for 15 seconds, 53° C. for 30 seconds, and 72° C for 30 seconds)are performed in an 7700 Prism spectrofluorometric thermal cycler(Applied Biosystems). For quantitative measurements, duplicates ofsix-fold serial dilutions (10⁶ to 10 copies) of RNA standards are usedas quantitative controls along with the samples being tested for a givenexperimental run. Viral RNA copy number for each clinical sample iscalculated by interpolation of the experimentally determined thresholdcycle for the test specimen onto a standard regression curve obtainedfrom the control RNA standards (the logarithm of the number of genomiccopies present in the clinical sample is inversely proportional to theobserved threshold cycle).

Explanation of how the invention solves the problems of the prior art.

The main problem with the prior art is its failure to consider theintrinsically polymorphic nature of coronaviruses and to account for thepossibility of continuous and/or multiple introductions of nonnon-genetically identical SARS-CoV strains into the human population.The present invention solves these problems by using multiple geneticsequences as targets (the S, E, M, and N genes). The primary principleof using four different genetic targets to identify SARS-CoV is to evadethe fundamental genetic drift of the virus. Thus, by using fourmolecular beacons, each specific for a different amplified SARS-CoVtarget, and each labeled with same colored fluorophore, the likelihoodof not detecting SARS-CoV due to continuous evolutionary changes in thevirus is minimized. Furthermore, the assay design includes the presenceof an internal positive control RNA, the reverse transcription and PCRamplification of which generates a signal in a different color, thusassuring that if there is an unexpected problem with RNA isolation,reverse transcription, or target amplification, the absence of thecontrol signal will indicate that a problem occurred.

Summary of the features of other SARS nucleic acid assays anddescription of the differences between those assays and the currentinvention. As outlined in the previous sections, there are five SARSnucleic acid assays: an RT-PCR assay published by Perris et al. [2], anRT-real-time-PCR assay published by Ksiazek et al. [1], a similarRT-real-time-PCR assay published by Drosten et al. [4], anotherRT-real-time-PCR assay published by Poon et al. [7], and a PCR-basedSARS diagnostic assay developed by Lipkin and Briese—no publication isavailable describing this assay. All of the published assays use RNAextracted from clinical samples. In addition, all of the publishedassays initiate reverse transcription of SARS-CoV RNA with randomprimers, and all of the published assays generate cDNA with primerspreviously designed to enable the amplification of conserved regions ofopen reading frame 1b (ORF1b), in order to achieve broad reactivity withthe coronavirus genus. The present invention differs from theseinventions in many different ways. The present assay uses four differenttargets instead of one and it has an internal positive RNA control(artificial RNA molecule) that can be reverse-transcribed and amplifiedwith primers designed for the viral genes (it possesses a unique targetrecognition sequence for detection by a unique molecular beacon). ThisRNA molecule serves as a control for RNA isolation, reversetranscription, and PCR amplification. The present uses real-time PCR fornucleic acid amplification and molecular beacons for real-timedetection. The present employs a dual-color detection scheme, a yellowsignal (tetrachlorofluorescein) for all four SARS-CoV targets and agreen signal (fluorescein) indicating that the internal positive controlhas been isolated, reverse transcribed, and PCR amplified.

Another important aspect of our invention, which is not an aspect of thepublished assays, is the construction of four viral RNA targets thatcontain gene-specific reverse transcription sequences built in to their3′ ends. Collectively, these molecules serve as SARS-specific positivecontrols. All of the present designs are thermodynamically compatible towork together in a five-amplicon multiplex assay.

Also multiplex polymerase chain reaction (mPCR) are envisioned. It is aprocedure for simultaneously performing PCR on greater than twodifferent sequences. A mPCR reaction comprises: treating said extractedDNA to form single stranded complementary strands, adding a plurality oflabelled paired oligonucleotide primers, each paired primer specific fora different short tandem repeat sequence, one primer of each pairsubstantially complementary to a part of the sequence in the sensestrand and the other primer of each pair substantially complementary toa different part of the same sequence in the complementary antisensestrand, annealing the plurality of paired primers to their complementarysequences, simultaneously extending said plurality of annealed primersfrom the 3′ terminus of each primer to synthesize an extension productcomplementary to the strands annealed to each primer, said extensionproducts, after separation from their complement, serving as templatesfor the synthesis of an extension product for the other primer of eachpair, separating said extension products from said templates to producesingle stranded molecules, amplifying said single stranded molecules byrepeating at least once said annealing, extending and separating steps.

Lower stringent conditions are routinely used to accommodate the captureof multiple target sequences that contain variations in their nucleicacid sequences. The stringency is reduced by either powering thetemperature of hybridization and wash or by modification of the buffer.When the stringent conditions are reduced and the target nucleic acidsequence is very similar to nucleic acid sequences of another genusspecificity of the capture probe for the target genus can be lost.

When multiple capture probes are used and are selected to compatible tovariations in the target nucleic acid sequences, the specificity underhigh stringent conditions can be regained. The blending of multipleprobes permits a single positive response for the presence of a group oftarget organisms.

In a multiplex assay, numerous conditions of interest are simultaneouslyexamined. Multiplex analysis relies on the ability to sort samplecomponents or the data associated therewith, during or after the assayis completed.

EXAMPLES Example 1 Design of SARS-CoV-Specific Molecular Beacons, andPrimers for Reverse Transcription and for PCR

Purpose: The overall rationale in the design of molecular beacons andoligonucleotides for our SARS assay is to construct mismatch-tolerantmolecular beacons that are thermodynamically compatible to work in afive-amplicon multiplex assay.

Design: The molecular beacons were designed so that they are able tohybridize to their targets at the annealing temperature of the PCR,while unbound molecular beacons remain in the closed conformation. Thesebasic aspects were achieved by using coronavirus gene-specific multiplealignments and thermodynamic considerations to select the targetsequences, the identity and length of the PCR primers, the identity andlength of the probe sequences (target recognition sequences), and thelength of the arm sequences.

Materials: In order to theoretically calculate the melting temperaturesof the PCR primers and the probe-target hybrids, was used the OligoToolkit that is available on the internet. The melting temperatures andsecondary structure predictions of the molecular beacons were calculatedby using the DNA folding program developed by Michael Zuker that isavailable on the internet.

Results: The theoretical melting temperatures of the PCR primers wasabout 60° C.; the T_(m) of the reverse transcription primers was 47° C.;the T_(m) of the probe-target hybrid was about 63° C.; and the T_(m) ofthe stem hybrids of the molecular beacons was about 70° C.

Conclusion: The mismatch-tolerant molecular beacons that arethermodynamically compatible are designed to work in a finalfive-amplicon multiplex SARS-CoV assay.

See the following Figures: The molecular designs for the S gene targetof the SARS-CoV assay are described in detail in FIGS. 1-4; for the Egene target see FIGS. 5-8; for the M gene target, see FIGS. 9-12; forthe N gene target, see FIGS. 13-16; and for the Internal PositiveControl (IPC), see FIGS. 17 and 18.

Example 2 Experimental Characterization of the Molecular Beacons and theMolecular Beacon-target Complexes

Purpose: The overall rationale of these experiments is to evaluate thethermodynamic properties of the constructed molecular beacons prior tocarrying out real-time PCR experiments.

Design: For each molecular beacon, we have determined two meltingcurves—one for beacon alone, and one for the beacon-target complex—byusing the ABI Prism 7700 spectrofluorometric thermal cycler.

Materials: For each molecular beacon, melting curves were obtained bypreparing two tubes containing 50 μL of 200 nM molecular beacondissolved in 3.5 mM MgCl₂ and 10 mM Tris-HCl, pH 8.0, and by adding acomplementary oligonucleotide target to one of the tubes at a finalconcentration of 400 nM.

The fluorescence of each solution was determined as a function oftemperature, using a thermal cycler with the capacity to monitorfluorescence. Temperature was decreased linearly with time from 80° C.to 10° C. in 1° C. steps; with each holding period lasting one minute,and fluorescence intensity was measured during each hold.

Results: The theoretical melting temperature of the PCR primers was 60°C. ±2° C.; the theoretical T_(m) of the reverse transcription primerswas 47±2° C.; the theoretical T_(m) of the probe target-hybrids wasabout 63±3° C.; and the theoretical T_(m) of the stem hybrid of themolecular beacons was 70±2° C.

Conclusion: The designed mismatch-tolerant molecular beacons wasdetermined to correctly recognize their DNA targets, and to bethermodynamically compatible to work together in a five-ampliconmultiplex SARS-CoV assay.

Figures: The melting curves of the molecular beacons and the molecularbeacon-target hybrids are shown in FIG. 19.

Example 3 Uniplex SARS-CoV Viral and IPC PCR Amplifications, Using SYBRGreen to Detect the Amplicons

Purpose: The overall rationale of these experiments is to evaluate thePCR primers and PCR conditions.

Design: For each SARS-CoV gene-specific and IPC amplification, asynthetic target DNA was used. PCR reactions were performed using aspectrofluorometric thermal cycler (Cepheid).

Materials: The PCR protocols are shown in the following exhibits:

for S Gene:

(A) SYBR Green-based Detection of S Gene Amplicon (LK250) ofSARS-associated CoV

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μl LK251(10 pmole/μl) 0.5 μl LK252 (10 pmole/μl) 0.5 μl Sybr Green DNA (25X) 1.0μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(B) Molecular-beacon-based Detection of S Gene Amplicon (LK250) ofSARS-associated CoV

Mixture Per reaction dH₂0 15.75 μl 10X PCR Buffer (10X)  2.5 μl MgCl₂(25 mM)  4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹)  0.3 μl dNTP (25 mM)  0.3 μlLK251 (10 pmole/μl)  0.5 μl LK252 (10 pmole/μl)  0.5 μl LK249 Beacon (10pmole/μl)  0.25 μl Target DNA  1.0 μl TOTAL  25.0 μl

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(C) SYBR Green-based Detection of S Gene Amplicon (T7- and RT-Amplicon)

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μlLK251-T7 (10 pmole/μl) 0.5 μl LK252-RT (10 pmole/μl) 0.5 μl Sybr GreenDNA (25X) 1.0 μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 secfor the E Gene:(A) SYBR Green-based Detection of E Gene Amplicon (LK254) ofSARS-associated

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μl LK255(10 pmole/μl) 0.5 μl LK256 (10 pmole/μl) 0.5 μl Sybr Green DNA (25X) 1.0μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(B) Molecular-beacon-based Detection of E Gene Amplicon (LK254) ofSARS-associated CoV

Mixture Per reaction dH₂0 15.75 μl 10X PCR Buffer (10X)  2.5 μl MgCl₂(25 mM)  4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹)  0.3 μl dNTP (25 mM)  0.3 μlLK255 (10 pmole/μl)  0.5 μl LK256 (10 pmole/μl)  0.5 μl LK253 Beacon (10pmole/μl)  0.25 μl Target DNA  1.0 μl TOTAL  25.0 μl

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(C) SYBR Green-based Detection of E Gene Amplicon (T7- and RT-Amplicon)

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μlLK255-T7 (10 pmole/μl) 0.5 μl LK256-RT (10 pmole/μl) 0.5 μl Sybr GreenDNA (25X) 1.0 μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 secfor the M Gene(A) SYBR Green-based Detection of M Gene Amplicon (LK258) ofSARS-associated CoV

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μl LK259(10 pmole/μl) 0.5 μl LK260 (10 pmole/μl) 0.5 μl Sybr Green DNA (25X) 1.0μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(B) Molecular-beacon-based Detection of M Gene Amplicon (LK258) ofSARS-associated CoV

Mixture Per reaction dH₂0 15.75 μl 10X PCR Buffer (10X)  2.5 μl MgCl₂(25 mM)  4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹)  0.3 μl dNTP (25 mM)  0.3 μlLK259 (10 pmole/μl)  0.5 μl LK260 (10 pmole/μl)  0.5 μl LK257 Beacon (10pmole/μl)  0.25 μl Target DNA  1.0 μl TOTAL  25.0 μl

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(C) SYBR Green-based Detection of M Gene Amplicon (T7- and RT-Amplicon)

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μlLK259-T7 (10 pmole/μl) 0.5 μl LK260-RT (10 pmole/μl) 0.5 μl Sybr GreenDNA (25X) 1.0 μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 secfor the N Gene:(A) SYBR Green-based Detection of N Gene Amplicon (LK262) ofSARS-associated CoV

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μl LK263(10 pmole/μl) 0.5 μl LK264 (10 pmole/μl) 0.5 μl Sybr Green DNA (25X) 1.0μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(B) Molecular-beacon-based Detection of N Gene Amplicon (LK262) ofSARS-associated CoV

Mixture Per reaction dH₂0 15.75 μl 10X PCR Buffer (10X)  2.5 μl MgCl₂(25 mM)  4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹)  0.3 μl dNTP (25 mM)  0.3 μlLK263 (10 pmole/μl)  0.5 μl LK264 (10 pmole/μl)  0.5 μl LK261 Beacon (10pmole/μl)  0.25 μl Target DNA  1.0 μl TOTAL  25.0 μl

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(C) SYBR Green-based Detection of N Gene Amplicon (T7- and RT-Amplicon)

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μlLK263-T7 (10 pmole/μl) 0.5 μl LK264-RT (10 pmole/μl) 0.5 μl Sybr GreenDNA (25X) 1.0 μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 secand for Internal Positive Control (IPC)(A) SYBR Green-based Detection of Internal Positive Control (LK266)

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μl LK251(10 pmole/μl) 0.5 μl LK256 (10 pmole/μl) 0.5 μl Sybr Green DNA (25X) 1.0μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(B) Molecular-beacon-based Detection of Internal Positive Control(LK266)

Mixture Per reaction dH₂0 15.75 μl 10X PCR Buffer (10X)  2.5 μl MgCl₂(25 mM)  4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹)  0.3 μl dNTP (25 mM)  0.3 μlLK251 (10 pmole/μl)  0.5 μl LK256 (10 pmole/μl)  0.5 μl LK265 Beacon (10pmole/μl)  0.25 μl Target DNA  1.0 μl TOTAL  25.0 μl

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec(C) SYBR Green-based Detection of Internal Positive Control (T7- andRT-Amplicon)

Mixture Per reaction dH₂0  15 μl 10X PCR Buffer (10X) 2.5 μl MgCl₂ (25mM) 4.0 μl Plat Taq DNA Pol (5 Uμl⁻¹) 0.3 μl dNTP (25 mM) 0.3 μlLK251-T7 (10 pmole/μl) 0.5 μl LK256-RT (10 pmole/μl) 0.5 μl Sybr GreenDNA (25X) 1.0 μl Target DNA 1.0 μl TOTAL 25.0 μl 

Smart Cycler (Cepheid)

DNA Denaturation & Enzyme Activation

Cycles: 1 Target Temperature (° C.): 95 120 sec

Primary Amplification

Cycles: 35 Denaturation: 95° C. 15 sec Annealing: 53° C. 15 sec SpectraON Extension: 72° C. 15 sec

Results: Real-time PCR amplification curves were consistent with thepresence or absence of target DNA and the amplicons that weresynthesized in the PCR reactions had the correct lengths.

Conclusion: The synthesized primers work well under uniform PCRconditions.

Example 4 Uniplex SARS-CoV Viral and IPC PCR Amplifications, UsingGene-specific Molecular Beacons

Purpose: The overall rationale of these experiments is to evaluate themolecular beacons, using the uniform PCR condition established in thetests with the PCR primers.

Design: For each SARS-CoV gene-specific and IPC amplification, asynthetic target DNA and molecular beacon are used. PCR reactions wereperformed using the spectrofluorometric thermal cycler (Cepheid). Foreach assay, utilized target dilutions are performed to establish thelinearity and the dynamic range of the molecular beacon-based real-timePCR assays.

Materials: The PCR protocols are shown above in Example 3 for the SGene, E gene, M Gene, N Gene, and the Internal Positive Control (IPC).

Results: Real-time PCR amplification curves were consistent with thepresence or absence of target DNA.

Conclusion: The SARS-CoV-specific and IPC-specific molecular beaconswork well in under uniform PCR conditions. See FIG. 20 Exhibit 25).

Example 5 Uniplex SARS-CoV Viral and IPC PCR Amplifications UsingBacteriophage T7 RNA Polymerase and Reverse Transcriptase Target PCRPrimers

Purpose: The overall rationale of these experiments is for each of thefive amplicons (SARS-CoV S, E, M, and N genes and IPC) to produce dsDNAmolecules that contain a bacteriophage T7 promoter target recognitionsequence at their 5′ ends and the gene-specific reverse transcriptaseprimer-binding site at their 3′ ends. The synthesized amplicons are usedfor in vitro RNA production. The synthesized RNA molecules have thespecific reverse transcriptase primer-binding site at their 3′ ends.

Design: For each SARS-CoV gene-specific and IPC amplification, asynthetic target DNA and bacteriophage T7-RT-PCR primers are used. PCRreactions were performed using the spectrofluorometric thermal cycler(Cepheid), using SYBR green to detect the amplicons.

Materials: The PCR protocols are shown in Example 3.

Results: Real-time PCR amplification curves are consistent with thepresence or absence of target DNA and the amplicons synthesized in thePCR reactions had the correct lengths.

Conclusion: SARS-CoV-specific bacteriophage T7-RT amplicons weregenerated for use as templates for in vitro RNA transcription, in orderto produce the target RNAs that will be used to qualify the PCR assays.See FIG. 20.

Example 6 In vitro RNA Transcription of SARS-CoV- and IPC-specific RNAMolecules

Purpose: The overall rationale for these experiments is to produce RNAmolecules containing the specific reverse transaction primer-bindingsite at their 3′ ends.

Design: For each SARS-CoV gene-specific and IPC amplification, aT7-RT-PCR dsDNA amplicon (generated from Experiment 5) is used.

Materials: RNA transcripts corresponding to the four SARS-CoV-specificand IPC alleles were prepared by in vitro transcription of PCR productsthat contain the T7 RNA polymerase promoter site, by using a MEGAscriptT7 kit (Ambion, Houston, Tex.).

Results: RNA molecules had the correct lengths, based on 8%polyacrylamide gel electrophoretic analysis of the product strands.

Conclusion: SARS-CoV-specific T7-RT amplicons were generated to be usedfor in vitro RNA transcription.

Example 7 Uniplex SARS-CoV-specific and IPC-specific RNA ReverseTranscription and Molecular Beacon-based Real-time PCR

Purpose: The overall rationale of these experiments is to test whetherthe RNA molecules containing the specific reverse transcriptionprimer-binding site at their 3′ ends can be reverse transcribed and thegenerated cDNA can be amplified and detected by real-time PCR.

Design: For each SARS-CoV gene-specific and IPC amplification, aT7-RT-PCR dsDNA amplicon (generated from Experiment 5) is used.

Materials: Reverse transcription with viral and IPC RNA was performed byadding 20 μL of SARS-CoV-specific and IPC-specific RNA to 0.125 μM ofSARS-CoV-specific and IPC-specific primers and incubated at 80° C. for 5minutes. The PCR-tubes were then immediately placed on ice for at least2 minutes. the reverse transcriptase reactions contained PCR buffer II(Applied Biosystems), 3 mM MgCl₂, 0.5 mM of each dNTP (GibcoBRL), 10 mMdithiothreitol, 20 Units ribonuclease inhibitor (Roche MolecularBiochemicals) and 80 Units SuperScript II RNase H-reverse transcriptase(GibcoBRL) in a final volume of 40 μL. The reactions were incubated at42° C. for 50 minutes, followed by inactivation at 70° C. for 15minutes. Real-time PCR of cDNA, using 10 μL cDNA, 1× PCR buffer, 3.5 mMMgCl₂, 0.5 mM of each dNTP, 0.4 μM of each molecular beacon, 0.4 μM ofeach PCR primer, and 2.5 Units AmpliTaq Gold DNA polymerase in a finalvolume of 50 μL. 35 cycles of amplification (94° C. for 15 sec, 53° C.for 30 sec, and 72° C. for 30 sec) were performed in a 7700 Prismspectrofluorometric thermal cycler (Applied Biosystems).

Results: SARS-CoV-specific and IPC-specific RNA molecules can be reversetranscribed, and the generated cDNA can be amplified and detected bySARS-CoV-specific and IPC-specific molecular-beacons in real-time PCRassays.

Conclusion: SARS-CoV-specific RNA molecules can be used as controls inthe final assay.

Example 8 Multiplex SARS-CoV-specific and IPC-specific RNA ReverseTranscription and Molecular Beacon-based Real-time PCR

Purpose: The overall rationale of this experiment is to multiplex theuniplex SARS-CoV-specific and IPC-specific RT-PCR reactions in amultiplex RT-PCR reaction.

Design: All five RT-PCR reactions are incorporated into one.

Materials: Identical to Experiment 7, with the exception that all fiveRT primers, four sets of PCR primers, and five molecular beacons areused in a single RT-PCR reaction

Results: SARS-CoV-specific and IPC-specific RNA molecules can be reversetranscribed and generate cDNA, and can then be amplified and detected bySARS-CoV-specific and IPC-specific molecular beacons in a real-time PCRassay.

Conclusion: SARS-CoV-specific RNA molecules can be used as controls in amultiplex assay.

Example 9 Evaluate the Complete SARS-CoV Assay Using Clinical Samples

Purpose: The overall rationale of these experiments is to evaluate theSARS-CoV assay using real samples (isolated SARS-CoV from cultures,primary patient isolates from saliva, and whole blood and stoolspecimens).

Design: Identical to Experiment 8.

Materials: Identical to Experiment 8.

Results: SARS-CoV assay detects SARS-CoV and discriminates betweenSARS-CoV and other non-pathogenic coronaviruses.

Conclusion: SARS-CoV assay detects SARS RNA extracted from cultured SARSstrains and primary isolates.

Example 10 Clinical Evaluation

64 samples collected from 23 individuals who were clinically diagnosedto have SARS based on CDC, WHO and Health Canada case definitions.

The sample types included: bronchial lavage and sputnum (pre-mortemsamples) as well as lung, liver, small and large bowel, and spleentissue (post mortem).

65 samples from 15 patients served as controls. 26 were bone marrowsamples sent for routing pathogen screening. 39 were post mortem organsamples from 10 patients who died during the outbreak but whose deathswere attributed to other causes including congestive heart failure,cerebrovascular accidents, atherosclerotic heart disease, chronicobstructive pulmonary disease, invasive Group A streptococcal infection,amiodorone pulmonary toxicity, and pulmonary fibrosis.

All samples were blindl assayed by using the developed assay (using allfour genes: S, E, M, N) and the specificity was 100%.

-   1. Ksiazek, T. G., et al. A novel coronavirus associated with severe    acute respiratory syndrome, N. Engl J Med, 2003, 348(20): p.    1953-66.-   2. Peiris, J. S., et al., Coronavirus as a possible cause of severe    acute respiratory syndrome, Lancet, 2003, 361 (9366): p, 1319-25.-   3. Munch, R., Robert Koch. Microbes Infect, 2003. 5(1): p. 69-74.-   4. Drosten, C., et al., Identification of a novel coronavirus in    patients with severe acute respiratory syndrome, N Engl J Med, 2003,    348(20): p. 1967-76.-   5. Marra, M. A., et al., The Genome sequence of the SARS-associated    coronavirus, Science, 2003, 300(5624): p. 1399-404.-   6. Rota, P. A., et al., Characterization of a novel coronavirus    associated with severe acute respiratory syndrome, Science, 2003,    300(5624): p. 1394-9.-   7. Poon, L. L., et al., Rapid diagnosis of a coronavirus associated    with severe acute respiratory syndrome (SARS), Clin Chem, 2003,    49(^Pt 1): p. 953-5.-   8. Penaert, M., P. Callebaut, and J. Vergote, Isolation of a porcine    respiratory, non-enteric coronavirus related to transmissible    gastroenteritis, Vet. Q. 1986. 8(3): p. 257-61.-   9. Ruan, Y. J., et al., Comparative full-length genome sequence    analysis of 14 SARS coronavirus isolates and common mutations    associated with putative origins of infection Lancet, 2003,    31(9371): p. 1779-85.

The content of each of the document identified above and cited in thespecification documents is expressly incorporated herein by reference.

1. A molecular-beacon-based multi-allelic real-time reversetranscription polymerase chain reaction (RT-PCR) multiplex assay for aSever Acute Respiratory Syndrome (SARS) virus comprising: (1) obtaininga sample; (2) isolating RNA from the sample; (3) placing the isolatedRNA in a tube along with primers specific for spike (S), envelope (E),membrane (M), and nucleocapsid (N) genes of the SARS virus, wherein theS primer includes SEQ ID NO:18, the E primer includes SEQ ID NO:38, theM primer includes SEQ ID NO:59, and the N primer includes SEQ ID NO:80,(4) reverse transcribing the isolated RNA in the presence of a reversetranscriptase to form a cDNA unique for each of the S, E, M and N SARSviral genes, (5) real-time amplifying the SARS cDNA in the presence offour distinct types of molecular beacons, each labeled with the samefluorophore and specific for a different SARS coronavirus (CoV) geneselected from S, E, M and N viral genes, and four S, E, M and Ngene-specific primer pairs, wherein the molecular beacons are: for Sgene: LK249 5′-FAM-CCCACGCCAGAAGGTAGATCACGAACTACACGTGGG-3′-Dubcyl (SEQID NO:14); for E gene: LK2535′-FAM-CCTCCGCACGAAAGCAAGAAAAAGAAGTACGCCGGAGG-3′-Dubcyl (SEQ ID NO:34);for M gene: LK2575′-FAM-CCTCCGACCCAATTAATTCTGTAGACAGCAGCCGGAGG-3′-Dubcyl (SEQ ID NO:55);and for N gene: LK2615′-FAM-CCTCCGTACCATCTGGGGCTGAGCTCTTFCATCGGAGG-3′-Dubcyl (SEQ ID NO:76);wherein the S, E, M and N gene-specific primer pairs are: for S geneLK251 5′-CTCTATGTTTATAAGGGCTATCAACC-3′ (SEQ ID NO:16) LK2525′-CCAAGAGGCAACTTAAAAATAGGTTTC-3′ (SEQ ID NO:17); for E gene LK2555′-CGGAAGAAACAGGTACGTTAATAG-3′ (SEQ ID NO:36) LK2565′-AAGCGCAGTAAGGATGGCTA-3′ (SEQ ID NO:37); for M gene LK2595′-CTTGTTTTCCTCTGGCTCTTG-3′ (SEQ ID NO:57) LK2605′-CAAGCCATTGCAATCGCAATC-3′ (SEQ ID NO:58); and for N gene LK2635′-ACGAGTTCGTGGTGGTGAC-3′ (SEQ ID NO:78) LK264 5′-CGTAGGGAAGTGAAGCTTC-3′(SEQ ID NO:79); and (6) measuring fluorescence, which is a collectivemeasure of the presence of the SARS virus.
 2. The assay of claim 1wherein the SARS virus is SARS-CoV.
 3. The assay of claim 1, whereinstep (3) further comprises a RNA internal positive control (IPC) andstep (5) further comprises IPC-specific primers and a fifth molecularbeacon that is specific for the IPC and labeled with a differentfluorophore, which can be distinguished from the SARS-CoV gene-specificmolecular beacon fluorophore, and wherein step (6) further includes ameasurement of fluorescence at a wavelength unique for the fifthmolecular beacon.
 4. The assay of claim 1 wherein the sample sourceincludes blood, tissue, sputum, or nasopharyngeal aspirations.
 5. Theassay of claim 1 wherein the measuring step includes simultaneousautomated amplicon detection in a spectrofluorometric thermal cyclerthat measures the fluorescence intensity of each color during theannealing phase of each thermal cycle.
 6. The assay of claim 3 whereinthe IPC-specific primers are included in step (5) and have the followingsequences: SEQ ID NO:16 and SEQ ID NO:37.